Water plays a central role in the stability, dynamics, and function of biomolecules (Hummer, Mol. Phys. 2007). Through the hydrophobic effect and hydrogen bond interactions, water is a major factor in the folding of proteins. In many enzymes, it participates directly in the catalytic function. In particular, water in the protein interior often mediates the transfer of protons between the solvent medium and the active site. Such water, often confined into relatively nonpolar pores and cavities of nanoscopic dimensions, exhibits highly unusual properties, such as high water mobility, high proton conductivity, or sharp transitions between filled and empty states. Proteins exploit these unusual properties of confined water in their biological function, e.g., to ensure rapid water flow in aquaporins, or to gate proton flow in proton pumps and enzymes.[unreadable] [unreadable] In collaboration with the experimental groups of Prof Sol Gruner (Cornell) and Prof Brian Matthews (University of Oregon and HHMI), we have shown that water filling of a large cavity in a T4 lysozyme mutant is highly sensitive to the solvent conditions (Collins et al., J. Mol. Biol. 2007). By combining high-pressure X-ray crystallography and molecular dynamics simulations, we found that application of modest pressure causes approximately four water molecules to enter the cavity while the protein itself remains essentially unchanged. Under pressure, the cavity remained rigid, while other regions of the protein deform substantially. The resultant picture of the protein interior is one in which conformationally fluctuating side groups provide a liquid-like environment, that makes water penetration feasible both kinetically and thermodynamically.[unreadable] [unreadable] In collaboration with Prof Jay Rasaiah (University of Maine), we studied the interior hydration in tetrabrachion, a hyperstable protein of the surface layer of a deep-sea organism (Yin et al., J. Am. Chem. Soc. 2007). This highly unusual protein exhibits what may well be the largest nonpolar interior hole of any protein found so far. We showed that hydrogen-bonded water clusters of seven to nine water molecules are thermodynamically stable in this cavity at both ambient temperature and 365 K, the temperature of optimal growth. The stability, as measured by the transfer free energy of the optimal size cluster, decreases with increasing temperature. Water filling is thus driven by the energy of transfer and opposed by the transfer entropy, both depending only weakly on temperature. Our calculations suggest that cluster formation becomes unfavorable just slightly above the temperature of optimal growth of the organism. Drying of the cavity thus precedes protein denaturation. This observation led us to the hypothesis that the unusually large cavity in tetrabrachion may act as binding site for two proteases, shown to bind just above the large cavity, possibly explaining the unusual thermostability of the resulting protease-stalk complexes (up to 390 K, 120 deg-C).[unreadable] [unreadable] In collaboration with Prof Christoph Dellago (University of Vienna), we explored the transfer of protons across low-dielectric membranes, mediated by ordered chains of water molecules (Dellago and Hummer, Phys. Rev. Lett. 2006). Free energy and rate constant calculations show that protons move across the membrane diffusively along single-file chains of hydrogen-bonded water molecules. Proton passage through the membrane is opposed by a high barrier in the effective potential, reflecting the large electrostatic penalty for desolvation and reminiscent of charge exclusion in biological water channels. Our observations not only provide an explanation for the low rate of proton transfer trough aquaporin-type channels, but are also relevant for the design of novel proton membranes in fuel cells.